16s rDNA amplicon of mouse feces (GAN_group)
Eight-week-old C57BL/6 ApoE-/- female mice were randomly divided into five groups with eight mice in each group: (1) CON (control diet); (2) GC (Gubra Amylin NASH diet [GAN diet] + L-carnitine in water [1.3 %]); (3) GC+GEOLow (GC + Ginger essential oil (GEO) [50 mg/kg bw/day]) ; (4) GC+GEOHigh (GC + GEO [100 mg/kg bw/day]); (5) GC+CIT (GC + Citral [20 mg/kg bw/day]). The control diet contained 10 kcal% fat (Research Diets, Inc., NJ, USA; D12450K), and the GAN diet contained 40 kcal% fat, 20 kcal% fructose, and 2 % cholesterol (GAN diet; Research Diets, Inc., NJ, USA; D09100310). GEO and citral were dissolved in soybean oil. All mice were fed experimental diets and liquid ad libitum. After 16 weeks, the mice were sacrificed. Mouse fecal samples were removed from the colons during the sacrifices, snap-frozen using liquid N2, and stored at -80 C before use. Genomic DNA from the feces was extracted using the QIAmp Power Fecal Pro DNA Kit (QIAGEN, Netherlands) and quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). The V3-V4 hypervariable region of the 16S rRNA gene was amplified using the primer pair ((Forward=5-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3) and Reverse=5-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3)). Polymerase chain reaction (PCR) amplification was conducted in a 25 uL reaction mixture containing 5 ng of DNA template, 0.2 uM forward and reverse primers, and 12.5 uL of 2xTaq Master Mix (KAPA HiFi HotStart ReadyMix, Roche, Switzerland). The PCR conditions included an initial step at 95 C for 3 min, followed by 25 cycles of 95 C, 55 C, and 72 C for 30 s each, and a final extension at 72 C for 5 min. Amplified products were subsequently visualized by using 2% agarose gel electrophoresis. Dual index and Illumina sequencing adapters were joined using a Nextera XT Index Kit via PCR. PCR product cleanup was conducted using AMPure XP beads to purify amplicon. The sizes of PCR products were validated using the Bioanalyzer DNA 1000 chip. Library quantification was carried out for quality control before sequencing using the Agilent Technologies 2100 Bioanalyzer. The pooled libraries were subjected to paired-end sequencing (2x300 bps) using the Illumina MiSeq platform.